Epigenetic modifications of GABAergic

interneurons are associated with the

schizophrenia-like phenotype induced by prenatal
stress in mice
Francesco Matrisciano, Patricia Tueting, Ishani Dalal, Bashkim Kadriu Dennis R.
Grayson, John M. Davis, Ferdinando Nicoletti, Alessandro Guidotti

By Ellie Kalman
  Human studies suggest prenatal stressors
are related to high risk for cognitive and
behavioral abnormalities in psychiatric
illness (Markham and Koenig, 2011).
 Exposure of rats/mice to stresses,
immune challenges, infections and
Background: malnutrition during pregnancy leads to
Prenatal disruption of behavioral and
neurochemical parameters in adult
Sensitivity offspring that mimic aspects of major
neuropsychiatric disorders (Borrell et al., 2002;
Moreno et al., 2011; Fatemi et al., 2008; Fortier et al., 2007;
Kinnunen et al., 2003; Koenig et al., 2005; Winter et al., 2008;
Zuckerman and Weiner, 2005; Shi et al., 2003).
  Lack of definitive genetic abnormalities
causally related to psychotic disorders
implicates epigenetic factors (Petronis et al., 1999).
 DNA methyltransferases (DNMT1 and
DNMT3a) that put methyl groups onto the C-
5 position of promoter cytosines, are highly
Background: overexpressed in in GABAergic neurons of SZ
Role of and BP disorder patients (Veldic et al., 2005, 2007;
Ruzicka et al., 2007; Guidotti et al., 2011).
Epigenetics
 High levels of DNMT are expressed in neuro-
progenitor cells, and DNMT expression as
well as DNA methylation can be increased in
post-mitotic neurons by early-life stressors
(Murgatroyd et al., 2009; Meaney and Ferguson-Smith, 2010; Weaver
et al., 2007)
  Promoters of reelin, a glycoprotein
that regulates of corticogenesis and
neuronal cell positioning in the
prenatal period, are hyper methylated
 Promoters for GAD67 (glutamate
decarboxylase), an enzyme that
synthesizes GABA from glutamate
Background: throughout the neuron for activities
such as synaptogenesis and injury
Role of protection, are hyper methylated.
Epigenetics  Downregulation in the expression of
GABAergic genes associated with
DNMT overexpression in GABAergic
neurons has been regarded as a
characteristic phenotypic component
of the neuropathology of psychotic
disorders (Guidotti et al., 2011)
  So far, there is no evidence in mice regarding:
 The expression and function of DNMT in the
developing brain
 DNMT changes in response to prenatal stress
 The effect of epigenetic changes on behaviors
related to SZ
Rationale for
Experiment:  The authors of this paper sought to determine
whether adverse prenatal experiences could
provoke neuropsychiatric disturbances by
decreasing DNA methylation that prevents
the overexpression of DNMT in GABAergic
neurons
  If adverse prenatal experiences lead to
the epigenetic changes associated with
neuropsychiatric disturbances, then the
offspring of restraint stressed mothers
Hypothesis:
should show decreased DNA methylation
that prevents the overexpression of DNMT
in GABAergic neurons.
  Experimental group (PRS): pregnant mice
subjected to a restraint stress using a
Experimental
plastic tube (10 cm 3 cm) for 30 min twice
Design:
daily from embryonic day 7-21
Prenatal
Conditions  Control group: pregnant mice left
undisturbed throughout gestation
  RNA extracted from frontal cortex and
hippocampus
 Absolute amount of DNMT1 and 3a
Experimental expression measured by reverse
Design: transcription polymerase chain reactions
RNA Analysis (RT-PCR) followed by agarose gel
electrophoresis
 Results: Increased
expression of
DNMT1 and 3a

Figure 1. PRS causes an early and long lasting increase in the expression of DNMT1 and 3a in the mouse
frontal cortex and hippocampus. DNMT1 and 3a mRNA levels in the frontal cortex (FC) of control (Ctrl) and PRS
mice at PND 1, 7, 14, and 60 are shown in (A); levels in the hippocampus of Ctrl and PRS mice at PND 7 and 60
are shown in (B). NSE and GPDH were utilized as internal controls. p < 0.05.
  Western blot analysis: Frontal cortex
proteins DNMT1, reelin, and GAD67 were
incubated with primary antibodies, and
Experimental the primary antibodies were then
incubated with secondary anti- bodies.
Design: Samples were then imaged for relative
Protein Analysis densities.
 Compare levels of DNMT, reelin, and
GAD67 expression in PRS mice vs control
mice
 Results: PRS induced
changes in the DNMT1,
reelin, and GAD67 protein
levels in the mouse frontal
cortex

Figure 3: Immunoblot analysis shows an increase in the protein levels of DNMT1, and a marked decrease in reelin and GAD67 protein
levels in 60-day old PRS mice compared to controls. The representative immunoblots show a major band of 190 kDa for DNMT1, 160 kDa
for reelin, and 67 kDa for GAD67. All values are means ` S.E.M. of five mice (p < 0.05) vs. the corresponding Ctrl value. All data were
normalized by b-actin protein levels.
  Confocal fluorescence microscopy
in GAD67-GFP knock-in mice:
identify the location of DNMT with
Experimental respect to GABAergic neurons
Design:  Test localization of GAD67 and
Histology DNMT1 protein in cortical and
hippocampal GABAergic neurons
of 7-day old control and PRS mice
 Results: almost complete co-
localization of GAD67 and
PRS:
DNMT1 protein in cortical and
hippocampal GABAergic
neurons
Control:
 Figure 2: Confocal double
immunofluorescence labeling of
GFP/GAD67 (green), DNMT1
(red)
 DNMT1 is co-expressed with GFP
in corticolimbic GABAergic
neurons of the GAD67-GFP
knock-in mouse, and merged PRS:
images in orange (center panels).
 A) Frontal motor cortex layer II in
7 day old mice
 B) CA1 field of the hippocampus
in 7 day old mice
Control:
 Scale bars represent 20
micrometers.
  Test whether the overexpression of
DNMT1 in FC of PRS mice
correlates with an increased
binding of DNMT1 to specific reelin
Experimental
and GAD67 CpG-rich promoter
Design: Promoter
Analysis sequences
 Test whether the binding of DNMT1
correlates with changes in the
methylation of these promoters
  Chromatin immunoprecipitation assay:
measurements of DNMT1 and methyl CpG
binding protein 2 (MeCP2) binding to reelin
and GAD67 promoters
 MeCP2 binds to methylated DNA and helps form a
complex that turns off the gene

 DNA mixed with antibodies for DNMT1 and

Experimental MeCP2 which bind to those promoter regions
Design: that have been methylated
 Total DNA before immunoprecipitation (input) also
ChIP Assay measured

 Protein is digested by Proteinase K, and the

now protein free DNA collected
 PCR reaction to amplify and quantify %
methylated and immunoprecipitated DNA vs.
total input of DNA
 Results: PRS causes
an increase of
DNMT1 and MeCP2
binding to reelin and
GAD67 promoters

Figure. 4a. PRS causes an increase of: A) DNMT1 binding to specific reelin and GAD67
promoter regions in the FC of PND 60 mice; B) MeCP2 binding to the same reelin and GAD67
promoter regions in the FC of PND1 and PND 60 mice. Values are means S.E.M. of 6 mice (p < 0.05)
vs. the corresponding control (Ctrl) values (*) or vs. saline (#).
  Further testing whether the binding of DNMT1
correlates with changes in the methylation of reelin and
GAD67 promoters
 Methylated DNA immunoprecipitation: analyzed the
ratio of 5’ methylated cytosines (5MC) or 5’
hydroxymethylated cytosines (5HMC) to the
Experimental unmethylated cytosines of mouse reelin and GAD67
Design: CpG-enriched promoter fragments

MeDIP and  Used 5MC and 5HMC antibodies because bisulfite

conversion and most enzyme-dependent methods are
HMeDIP incapable of distinguishing 5MC from the
Promoter Analysis approximately 20% of methylcytosines in the brain that
are 5HMC which may have very different functions and
genomic locations
 Results: PRS causes an
increase of methylation
and hydroxymethylation
of cytosines in reelin and
GAD67 promoters

Figure 4b. PRS causes an increase of: C) 5-methylcytosine binding at reelin and GAD67
promoters in the FC of PND 60 mice, and D) 5- hydroxymethylcytosine binding at reelin and
GAD67 promoters in the FC of PND 60 mice. Values are means ` S.E.M. of 6 mice (p < 0.05) vs. the
corresponding control (Ctrl) values (*)
  Male offspring from each group exposed to an
unfamiliar, non-aggressive male mouse
 Measured locomotor activity, social interaction, and
prepulse inhibition at startling noise
 SZ associated with increased spontaneous locomotor
activity, social withdrawal, and deficits in attention
and information processing (more easily startled)

Experimental  Separate contextual fear conditioning experiment

trained mice to expect a foot shock in conjunction
Design: with a tone while in a specific training chamber
Behavior Analysis  Subsequent freezing behavior measured for several
days after the training where mice were placed in
the same chamber, but not delivered the tone and
shock.
 SZ associated with decreased ability to extinguish
conditioned fear behavior after threat no longer
present
 Results: PRS mice
significantly more
prone to stereotypical
SZ behavior

Figure 5. 60 PRS mice compared to controls were characterized by: (A) an increase in locomotor activity
(p=0.02); (B) a decrease in social interaction (p < 0.001); (C) a deficit in prepulse inhibition of startle (group
p=0.001, prepulse intensity p < 0.001); and (D) a deficit in contextual fear conditioning (freezing on re-
exposure to the context over the first three days following conditioning p=0.029)
  In drug administration experiments, mice were injected
with valproic acid, clozapine and then tested
 Valproic acid:
 Anticonvulsant because blocks voltage gated
sodium channels
 Inhibits both enzymes that degrade GABA and
receptors that reuptake GABA.
 Histone-deacetylase-inhibiting effects which
promotes more transcriptionally active chromatin
Experimental structures.

Design: Drug  Clozapine:

 Atypical antipsychotic that binds to both dopamine
Treatment and serotonin receptors.
 Antagonist at the 5-HT2A subunit of the serotonin
receptor, for which drugs like LSD are agonists
 Increased GABA neurotransmission
 Release of glutamate and release of NMDA
receptor agonists from astrocytes
 Reduces expression of astrocytic glutamate
transporters involved in reuptake
 Results: Schizophrenia-
like behavioral
abnormalities reversed
with antipsychotic drugs

Figure 7. SZ-like behavior in PRS mice reversed by treatment with valproic acid (70 mg/kg, i.p.; twice a day
for 5 days), or clozapine (5 mg/kg, s.c.; twice a day, for 5 days). Data of locomotor activity (A) p=0.001, social
interaction (B) p < 0.001, and Prepulse inhibition (C) p= 0.004, 24 h after the last drug injection, are shown.
Post-hoc comparisons indicated PRS veh mice differed from controls veh mice (*), and from PRS mice treated
with VPA (**) or clozapine (#).
  In another set of experiments, control and
PRS mice were injected with clozapine for
five days and then received a single injection
of MK-801 30 min before testing for
behavioral stereotypy
 Stereotypy, or persistent repetition of a
movement for no obvious purpose, is a
symptom of SZ
Extra Background
 MK-801 (a.k.a Dizocilpine) helps produce
on the Drugs: SZ both the positive and negative symptoms of
Worsening Drug SZ in animal models
 Noncompetitive antagonist of the NMDA
receptor
 Binds inside ion channel of receptor (in
several places where agonist PCP binds)
and prevents flow of ions through the
channel
 Results: MK-801
increases stereotypic
behavior in PRS mice,
and clozapine
decreases stereotypic
behavior in PRS mice
treated with MK-801

Figure 6. Increased stereotypic behavior in PRS mice induced by NMDA receptor blockade with a low
dose of MK-801. Among PRS mice not treated with clozapine, MK-801 significantly increased stereotypy
counts. Stereotypy counts in response to MK-801 within PRS group are significantly lower when paired
with clozapine treatment.
  In PRS mice, SZ-like behavior measured in the adult is
associated with a persistent upregulation of DNMT1/3a
in cortical and hippocampal GABAergic neurons
during neurodevelopment and by a sustained binding
of DNMT to GABAergic gene promoters
 Associated with hyper methylation of reelin, GAD67, and
Discussion: likely other GABAergic promoters

 Epigenetic phenotype present in cortex and

Evaluation of hippocampus of PRS mice is reminiscent of the
Mouse Model epigenetic phenotype shown in the brain of patients
with SZ and BP disorders
 PRS mice and post-mortem brain of SZ patients have in
common increased levels of DNMT
  SZ and BP post-mortem brains have GABAergic
neuropathology in prefrontal cortex and hippocampus
that differs from the non-psychotic brain
 Decreased GAD67, the rate limiting step enzyme in GABA
synthesis
 Decreased reelin, the extracellular matrix protein that
regulates dendritic spine maturation and glutamate
receptor structure and function
Discussion:  DNMT-induced GABAergic deficits could be the basis
Human Brains for the disturbance of reciprocal interactions between
GABAergic, glutamatergic, and monoaminergic
neurons in both human and mouse brains
 Likely explains exacerbation of psychotic episodes
elicited by NMDA receptor antagonists to SZ and BP
disorder patients
  Due to higher levels of DNMT in cortex and
hippocampus from birth to adulthood, we can infer that
in PRS mice the increase in DNMT is probably the result
of changes occurring during embryonic life
 Implicates stress during embryonic period, as DNMT
was elevated in PRS mice even on Day 1 before
Discussion: differences in maternal care could be a significant
Mouse Prenatal factor
Environment &  In Swiss mice, stressed mothers raising stressed pups
exhibit maternal care comparable to that of non-stressed
Maternal Care mothers raising non-stressed pups

 Restraint stress during pregnancy in mice leads to a

clozapine and valproic-acid-sensitive behavioral
psychosis-like phenotype in offspring
  Exposure of pregnant women to
psychological stress, malnutrition, or viral
infection during pregnancy is associated with
an increased incidence of psychosis later in
the life of offspring
 Neurochemical, behavioral, and
Discussion: pharmacological responses observed in adult
offspring of mothers exposed to stress during
Human Prenatal pregnancy appear to parallel some of the
Environment responses obtained in the adult onset of SZ
 Strongly supports the prenatal stress model in
mice as pertinent epigenetic animal model of
SZ
  Alterations in DNA methylation are
involved in the etiopathogenesis of
SZ
 A presumable mechanism by which
prenatal stress could disrupt GABA-
Discussion: glutamate interactions and increase
Implications stereotypy (induced by the NMDA
receptor antagonist MK-801) may
involve changes in DNA
methylation and the expression of
DNMT in corticolimbic GABAergic
neurons
  It is conceivable that
neurodevelopmental changes in
humans can occur as a response to
both pre and post-natal stressful
situations, thus preventing the normal
reduction in DNMT expression that
Discussion:
occurs with development
Limitations
 Cannot currently establish if a similar
time course in changes in DNMT occur
in the human brain
  Valproic and clozapine abolished the
hyperactivity, stereotypy, social
interaction, and PPI shown in PRS mice,
but had no observable effect in control
mice
Discussion:  Clozapine even blocked the increased
stereotyped behavior in PRS mice induced by
Moving low doses of MK-801
Forward  This suggests a specificity of action on the
epigenetic mechanisms that underlie the
behavioral pathology in PRS mice which
has yet to be studied
  Effects of prenatal stress on
behavior are indicative of
neocortical inhibitory/excitatory
circuit imbalance and changes in
the expression of DNMT, GAD67,
and reelin, suggesting that PRS
mice can be a valid animal model
Conclusions: to study the epigenetic mechanisms
underlying SZ and BP disorders
 PRS mice are suitable for validating
new compounds with potential
antipsychotic activity acting at an
epigenetic level